The invention relates to a method for the detection of an analyte in a competitive immunoassay in the presence of an analyte derivative, and to a kit for carrying out this detection method.
Immunological detection methods have gained great importance in in vitro diagnosis. The reason for this is that they are highly specific and extremely sensitive. Moreover, these assays are distinguished by simple handling. The detection methods are based on the immunological interaction between the analyte to be detected and its binding partner or partners.
In the case of sandwich assays, the analyte is bound like a sandwich by two different antibodies. One of the two antibodies carries a label (marker), whereby its concentration can be determined. In the case of small analytes, the sandwich method is excluded, as for steric reasons two different antibodies can not simultaneously bind to the analyte. Here, as a rule, competitive assays are used. In these, the analyte and a synthetic derivative of the analyte compete for the binding sites of the antibody. As a rule, either the analyte derivative (classic competitive method) or the antibody is labeled.
Often, the antibody is bound in the classic competitive method to a solid phase, while in the method using labeled antibody the analyte derivative is immobilized (solid phase antigen techniques, e.g. SPALT=solid phase antigen luminescence technique).
In the competitive assay method the methods using labeled antibodies have gained particular importance in the determination of the freely available portion of an analyte. “Freely available” here means that these analytes are not bound as ligands by their natural receptors, which can occur, for example in the serum. Methods of this type have gained importance, for example, in the determination of free triiodothyronine (FT3) and free thyroxine (FT4). In order to be able to determine the proportion of analyte independently of the concentration of binding proteins occurring in the sample (for example serum proteins), the analyte derivative must change its properties on binding to, solid phases in such a way that it can still interact with the antibody, but not—or only insignificantly—with the binding protein. A method of this type was described as a 1-step assay, for example, in EP-A-0 103 605.
This method using labeled antibodies, in particular, has the advantage of easy tracer preparation, as the labeling of an antibody is not a problem in most cases. It is therefore still to be preferred to the classical competitive method if an analyte tracer is available which shows the abovementioned, desired binding behavior (EP-A-0 026 103).
When carrying out the abovementioned method, the choice of an analyte derivative having suitable affinity for the antibody often causes problems. These result from the fact that the affinity of the. analyte derivative for the antibody for a meaningful assay result must be in a certain ratio to the affinity of the analyte for the antibody (cf. EP-A-0 254 929, EP-A-0 324 540, EP-A-0 303 284). If, for example, the affinity of the analyte derivative for the antibody is too high, the reaction equilibrium in the assay mixture will shift too much in the direction of the antibody-analyte derivative complex, which leads to a reduction in the meaningfulness of the assay. In order to remedy this disadvantage, a preincubation of the analyte with the antibody can be carried out (cf. EP-A-0 182 385). If, on the other hand, the affinity of the analyte derivative is too low, a preincubation of analyte derivative and antibody first can be of use. A disadvantage in both cases, however, is the necessity of having to carry out the assay in two steps. A possibility of circumventing this disadvantage consists in chemically modifying the analyte derivative in order to produce a suitable affinity for the antibody. However, a method of this type is also laborious and, with small analyte derivatives, often associated with difficulties or not possible to carry out at all.